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Description

The Vybrant™ FAM Poly Caspases Assay Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For poly caspases, this recognition sequence is valine-alanine-aspartic acid (VAD). A fluorescein group is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added. This kit includes the FAM-VAD-FMK FLICA reagent, Hoechst 33342 stain, and propidium iodide stain, which allows the simultaneous evaluation of caspase activation, membrane permeability, and cell cycle by flow cytometry.